A multiply antibiotic resistant strain of Streptococcus faecalis, LDR175, isolated from a broiler in North Carolina, was used as a plasmid donor in conjugation experiments. A 58 kilobase pair (kbp) plasmid, pLDR517, mediating resistance to erythromycin (Em), kanamycin (Km) and streptomycin (Sm), was mobilized into a S. faecalis recipient strain during these experiments. Purified pLDR517 DNA did not hybridize to any of the previously coned Em, Km or Sm resistance determinants from the well-characterized streptococcal R plasmid, pJH1. The resistance genes from pLDR517 are currently being cloned to determine the extent to which they share homology to DNA from other streptococci with the same resistance phenotype, also previously shown to be unrelated to DNA from pJH1. A unique spectinomycin (Sp) resistance determinant, mediating resistance (2,000 ug/ml) to Sp but not Sm, has been cloned from a plasmid, pDL55, originally from a human clinical isolate of S. faecalis, strain LDR55. The cloned determinant has been sequenced and contains a 783 base open reading frame encoding a protein with a predicted molecular size of 18,943 daltons. The Sp resistance gene shares approximately 48% base sequence homology with a similar gene encoded on a plasmid from Staphylococcus aureus, and the respective gene products share between 40 to 50% amino acid sequence homology. The streptococcal and staphylococcal genes are currently being used as hybridization probes with purified DNA from Neisseria gonorrhoeae isolates from patients in Korea expressing high level resistance to Sp but not Sm.